The present invention is generally directed to inhibiting or slowing blood coagulation, and more particularly to using lactadherin or a fragment thereof as an agent for inhibiting or slowing blood coagulation.
There are several anticoagulant drugs which are in widespread clinical use and many others in development, including clinical trials. However, none of these agents have the mechanism of blocking access of blood proteins to procoagulant membrane surfaces. Inhibition of coagulation at this stage is an earlier step than most anti-coagulants target and, it is specifically an anticoagulant step as opposed to a step which would inhibit platelet aggregation or adhesion.
Investigators have sought anticoagulants that would work via this mechanism for decades. Phospholipases from snake venoms can function as anticoagulants by competing for membrane binding sites and the function of several phospholipases have been analyzed in detail. These proteins are unsuitable for use in humans because the corresponding enzymatic activity (phospholipases cleave phospholipids releasing lysophospholipids and free fatty acids) cause inflammation and tissue injury. A single class of agents, annexins, have been shown to inhibit blood coagulation by blocking the membrane surface. These proteins have been evaluated as anticoagulants in animals. The efficacy is modest, at least partly because annexin is more fastidious in its requirements for membrane lipids than most blood clotting proteins. Thus, much of the procoagulant membrane of cells remains unblocked, even with a vast excess of annexin V. We compared the anticoagulant efficacy of annexin V directly to that of lactadherin.
Lactadherin is a MW 47,000 glycoprotein of milk fat globules. It has also been known as PAS-6/7, indicating the two glycosylation variants (Reference 1), bovine-associated mucoprotein, BA-46, P47, and MFG-E8 (Reference 2). Lactadherin has a domain structure of EGF1-EGF2-C1-C2 in which EGF indicates epidermal growth factor homology domains, and the C domains share homology with the discoidin family including the lipid-binding “C” domains of blood coagulation factor VIII and factor V (FIG. 1) (Reference 2). The second EGF domain displays an Arg-Gly-Asp motif (Reference 3) which binds to the αvβ5 and αvβ5 integrins (References 1 and 4-6). The second C domain binds to phospholipids (Reference 6).
In milk fat globules, lactadherin lines the surface of the phospholipid bilayer which surrounds the central triglyceride droplet, apparently stabilizing the bilayer (Reference 7). Lactadherin decreases the symptoms of rotavirus infection in infants, possibly by binding to rotavirus particles via carbohydrate moieties (Reference 8). In tissue sections, lactadherin is found localized on the apical portion of secretory epithelium in the breast (Reference 7). Abundant expression by breast carcinoma tissue makes lactadherin a potential target for antigen-guided radiation therapy (Reference 9). Lactadherin is also found on the apical surface of epithelia in the biliary tree, the pancreas, and sweat glands (Reference 7) and is synthesized by aortic medial smooth muscle cells (Reference 10). Function in these tissues remains unknown. Lactadherin has been identified as a zona pellucida-binding protein on the acrosomal cap of sperm (Reference 11).
Blood coagulation factor VIII and factor V bind to phospholipid membranes via “C” domains which share homology with lactadherin (References 12-14). Remarkable features of membrane binding for these proteins include high affinity (KD approx. 2 nM) (Reference 15) and sufficient specificity so that no plasma proteins compete for membrane binding sites (Reference 16). Factor VIII binds via stereo-selective interaction with the phospho-L-serine motif of phosphatidylserine (PS) (Reference 17). Factor V also exhibits stereoselective interaction with PS (Reference 18). Binding of factor VIII is enhanced by the presence of phosphatidylethanolamine (PE) in the membrane (Reference 19), by unsaturated phospholipid acyl chains (Reference 20), and by membrane curvature (Reference 19). The crystal structures of the C2 domains of factors VIII and V suggest that membrane binding is mediated by two pairs of hydrophobic residues displayed at the tips of β-hairpin turns (References 21-22). Mutagenesis studies have confirmed the role of these residues in phospholipid binding (Reference 23). The homology of the lactadherin C domains with those of factors VIII and V suggests that similar phospholipid binding properties may exist. Indeed, lactadherin has been found to bind selectively to PS (Reference 24) and to utilize primarily the C2 domain in its lipid binding (Reference 6).
Annexin V, like factor VIII and factor V, exhibits high affinity, PS-dependent membrane binding (Reference 25). However, the quadruplicate membrane-binding motifs of annexin V are not homologous with the discoidin-like domains of lactadherin, factor VIII, and factor V (Reference 26). Corresponding to the difference in structure, the membrane binding mechanism is different. In addition, annexin V requires Ca++ for membrane-binding and the binding is chiefly hydrophilic in nature (Reference 27). Annexin V does have the capacity to compete for a fraction of the phospholipid binding sites utilized by the factor VIIIa-factor IXa enzyme complex and the factor Xa-factor Va enzyme complex of the coagulation cascade so that it functions in vitro as a membrane-blocking anticoagulant (Reference 28). The well-defined membrane-binding and anti-coagulant properties of annexin V make studies with annexin V suitable controls for studies with lactadherin.